Fn secreted by CAFs promotes CAF–cancer cell association and directional cancer cell migration. (A) Time-lapse images showing co-culture of DU145 prostate cancer cells (red) with NFs (left; green) or CAFs (right; green) in microfluidic devices. Bar, 20 µm (Videos 1 and 2). (B) Schematic representation of the calculation to determine the association index between fibroblasts and cancer cells. (C) Association index of DU145 cells with NFs or CAFs. (D) Schematic representation for the calculation to determine the directionality ratio. (E) Directionality ratio of DU145 cells in co-cultures with NFs or CAFs. (C and E) The data represent ≥30 cells per condition in four individual experiments. (F) Fn staining of NF + DU145 cells (left) and CAF + DU145 (right) cell co-cultures. Fn, green; F-actin, phalloidin, red. Bars: (F, top) 20 µm; (bottom) 10 µm. White boxes indicate areas of increased magnification. (G) Co-culture of CAFs (unlabeled) with DU145 cells (red), cultured in culture medium supplemented with 5 µg/ml FITC–Fn (green). Bar, 20 µm. Arrows point to the leading edge of the cancer cell, where it binds to the Fn fibers assembled by CAFs (Video 3). (H) Time-lapse images showing co-culture of DU145 cells (red) with CAFs (green) transfected with nontargeting siRNA control (Ctrl CAF, left) or Fn siRNA (Fn-KD CAF, right). Arrows point to the cells of interest. Bar, 50 µm (Video 4). (I) Association index for DU145 cells with control (Ctrl) or Fn–KD CAFs. (J) Directionality ratio of DU145 cells in co-cultures with control (Ctrl) or Fn–KD CAFs. (I and J) The data represent ≥30 cells per condition in three individual experiments (C, E, I, and J) ***, P < 0.001, determined by Mann-Whitney U test. All box plots range from the 25th to the 75th percentiles; the central line indicates the median, and the whiskers range from the 5th to the 95th percentiles.